Shuanglin Hao

HSV-mediated expression of interleukin-4 in dorsal root ganglion neurons reduces neuropathic pain

BackgroundTo examine the role of inflammatory mediators in neuropathic pain, we used a replication-defective genomic herpes simplex virus (HSV)-based vector containing the coding sequence for the anti-inflammatory peptide interleukin (IL)-4 under the transcriptional control of the HSV ICP4 immediate early promoter, vector S4IL4, to express IL-4 in dorsal root ganglion (DRG) neurons in vivo.ResultsSubcutaneous inoculation of S4IL4 in the foot transduced lumbar DRG to produce IL-4. Transgene-mediated expression of IL-4 did not alter thermal latency or tactile threshold in normal animals, but inoculation of S4IL4 1 week after spinal nerve ligation (SNL) reduced mechanical allodynia and reversed thermal hyperalgesia resulting from SNL. Inoculation of S4IL4 1 week before SNL delayed the development of thermal hyperalgesia and tactile allodynia, but did not prevent the ultimate development of these manifestations of neuropathic pain. S4IL4 inoculation suppressed non-noxious-induced expression of c-Fos immunoreactivity in dorsal horn of spinal cord and reversed the upregulation of spinal IL-1β, PGE2, and phosphorylated-p38 MAP kinase, characteristic of neuropathic pain.ConclusionHSV-mediated expression of IL-4 effectively reduces the behavioral manifestations of neuropathic pain, and reverses some of the biochemical and histologic correlates of neuropathic pain at the spinal level.

Gene transfer of glutamic acid decarboxylase reduces neuropathic pain

We tested whether transfer of the gene coding for glutamic acid decarboxylase to dorsal root ganglion using a herpes simplex virus vector to achieve release of GABA in dorsal horn would attenuate nociception in this condition. Subcutaneous inoculation of a replication‐defective herpes simplex virus vector expressing glutamic acid decarboxylase (vector QHGAD67) 7 days after selective L5 spinal nerve ligation reversed mechanical allodynia and thermal hyperalgesia; the antiallodynic effect lasted 6 weeks and was reestablished by reinoculation. QHGAD67 inoculation also suppressed induction of c‐Fos and phosphorylated extracellular signal–regulated kinase 1 and 2 in the spinal cord. Ann Neurol 2005;57:914–918

Gene transfer to interfere with TNFα signaling in neuropathic pain

We examined the role of spinal tumor necrosis factor-alpha (TNFα) in neuropathic pain of peripheral nerve origin. Two weeks after selective L5 spinal nerve ligation (SNL), rats exhibiting mechanical allodynia and thermal hyperalgesia showed a marked increase in full-length membrane-associated TNFα (mTNFα) in the dorsal horn of spinal cord, in the absence of detectable soluble TNFα peptide. Local release of the soluble p55 TNF receptor, achieved by herpes simplex virus vector-based gene transfer to dorsal root ganglion, resulted in a reduction of mTNFα and concomitant reductions in interleukin-1β and phosphorylated p38 MAP kinase. Subcutaneous inoculation of soluble p55 TNF receptor expressing HSV vector into the plantar surface of the hind foot ipsilateral to the ligation 1 week before SNL delayed the development of both mechanical allodynia and thermal hyperalgesia; subcutaneous inoculation into the hind foot ipsilateral to the ligation 1 week after SNL resulted in a statistically significant reduction in mechanical allodynia and thermal hyperalgesia that was apparent 1 week after inoculation. These results suggest a novel ‘reverse signaling’ through glial mTNFα, which may be exploited to downregulate the neuroimmune reaction in spinal cord to reduce chronic neuropathic pain.

HSV-mediated gene transfer of the glial cell-derived neurotrophic factor provides an antiallodynic effect on neuropathic pain

Neuropathic pain is a difficult clinical problem that is often refractory to medical management. Glial-derived neurotrophic factor (GDNF) administered intrathecally has been shown to prevent or reduce pain in an animal model of neuropathic pain, but cannot be delivered in the required doses to treat human pain. We have previously demonstrated that peripheral subcutaneous inoculation of a replication-incompetent herpes simplex virus (HSV)-based vector can be used to transduce neurons of the dorsal root ganglion. To examine whether HSV-mediated expression of GDNF could be used to ameliorate neuropathic pain, we constructed a replication-incompetent HSV vector expressing GDNF. Subcutaneous inoculation of the vector 1 week after spinal nerve ligation resulted in a continuous antiallodynic effect that was maintained for 3-4 weeks. Reinoculation of the vector reestablished the antiallodynic effect with a magnitude that was at least equivalent to the initial effect. Vector-mediated GDNF expression blocked the nonnoxious touch-induced increase in c-fos expression in dorsal horn characteristic of the painful state. Gene transfer to produce a trophic factor offers a novel approach to the treatment of neuropathic pain that may be appropriate for human therapy.

HSV-mediated transfer of interleukin-10 reduces inflammatory pain through modulation of membrane tumor necrosis factor α in spinal cord microglia

To dissect the molecular basis of the neuroimmune response associated with the genesis of inflammatory (nociceptive) pain, we constructed a herpes simplex virus-based gene transfer vector to express the antiinflammatory cytokine interleukin-10 (IL-10), and used it to examine the effect of IL-10 expression in activated microglial cells in vitro, and in inflammatory pain in vivo. IL-10 reduced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and decreased the expression of full-length membrane spanning tumor necrosis factor-α (mTNFα) following lipopolysaccharide stimulation of microglia in vitro. IL-10 also reduced intracellular cleavage of mTNFα and release of the soluble cleavage product sTNFα. Similar effects on TNFα expression were observed when the cells were pretreated with a p38 MAPK inhibitor. In animals, injection of a dilute solution of formalin in the skin resulted in an increase in mTNFα in spinal dorsal horn, without detectable sTNFα. Local release of IL-10 achieved by gene transfer reduced the number of spontaneous flinches in the early and delayed phases of the formalin test of inflammatory pain. The effect of IL-10 on nocisponsive behavior correlated with a block in phosphorylation of p38 and reduced expression of 26 kDa mTNFα in spinal microglia. The results emphasize the key role played by membrane TNFα in the spinal neuroimmune response in pain caused by peripheral inflammation.

Glial TNFα in the spinal cord regulates neuropathic pain induced by HIV gp120 application in rats

BackgroundHIV-associated sensory neuropathy (HIV-SN) is one of the most common forms of peripheral neuropathy, affecting about 30% of people with acquired immune deficiency syndrome (AIDS). The symptoms of HIV-SN are dominated by neuropathic pain. Glia activation in the spinal cord has become an attractive target for attenuating chronic pain. This study will investigate the role of spinal TNFα released from glia in HIV-related neuropathic pain.ResultsPeripheral gp120 application into the rat sciatic nerve induced mechanical allodynia for more than 7 weeks, and upregulated the expression of spinal TNFα in the mRNA and the protein levels at 2 weeks after gp120 application. Spinal TNFα was colocalized with GFAP (a marker of astrocytes) and Iba1 (a marker of microglia) in immunostaining, suggesting that glia produce TNFα in the spinal cord in this model. Peripheral gp120 application also increased TNFα in the L4/5 DRG. Furthermore, intrathecal administration of TNFα siRNA or soluble TNF receptor reduced gp120 application-induced mechanical allodynia.ConclusionsOur results indicate that TNFα in the spinal cord and the DRG are involved in neuropathic pain, following the peripheral HIV gp120 application, and that blockade of the glial product TNFα reverses neuropathic pain induced by HIV gp120 application.

Engineering an endomorphin-2 gene for use in neuropathic pain therapy

Abstract Endomorphin‐2 (EM‐2) is a carboxy‐amidated tetrapeptide that binds the μ‐opioid receptor with high affinity and is analgesic in several animal models of pain. Endomorphin peptides have been isolated from bovine and human brain, but no DNA sequences corresponding to a potential preproendomorphin gene have been identified in human genome sequence databases. In this study we designed a tripartite synthetic gene to direct production, cleavage, and amidation of EM‐2, and placed the endomorphin gene expression cassette in a replication defective Herpes simplex virus (HSV) vector (vEM2). Biosynthesis of amidated endomorphin‐2 peptide was quantified by radioimmunoassay and the identity confirmed by mass spectroscopy following vEM2 transduction of cultured primary dorsal root ganglion neurons. Subcutaneous inoculation of vEM2 resulted in vector delivery to dorsal root ganglion where expression of EM‐2 peptide from the engineered gene was confirmed by ELISA. vEM2 delivery provided an analgesic effect in the spinal nerve ligation model of neuropathic pain measured by reduction of mechanical allodynia and thermal hyperalgesia. The analgesic effect of vEM2 was blocked by intrathecal delivery of the μ‐receptor antagonist CTOP. The gene construct design described represents a broadly useful platform for biosynthesis and delivery of carboxy‐amidated peptides for therapeutic and experimental purposes, and the results demonstrate that HSV‐gene transfer to sensory neurons provides an effective means to achieve local biosynthesis of endomorphin peptides for the treatment of chronic pain.

Effects of transgene-mediated endomorphin-2 in inflammatory pain

We examined the analgesic properties of endomorphin‐2 expressed in DRG neurons transduced with a non‐replicating herpes simplex virus (HSV)‐based vector containing a synthetic endomorphin‐2 gene construct. HSV‐mediated endomorphin‐2 expression reduced nocisponsive behaviors in response to mechanical and thermal stimuli after injection of complete Freunds adjuvant (CFA) into the paw, and reduced peripheral inflammation measured by paw swelling after injection of CFA. The analgesic effect of the vector was blocked by either intraperitoneal or intrathecal administration of naloxone methiodide, blocking peripheral and central μ opioid receptors, respectively. Endomorphin‐2 vector injection also reduced spontaneous pain‐related behaviors in the delayed phase of the formalin test and in both CFA and formalin models suppressed spinal c‐fos expression. The magnitude of the vector‐mediated analgesic effect on the delayed phase of the formalin test was similar in naïve animals and in animals with opiate tolerance induced by twice daily treatment with morphine, suggesting that there was no cross‐tolerance between vector‐mediated endomorphin‐2 and morphine. These results suggest that transgene‐mediated expression of endomorphin‐2 in transduced DRG neurons in vivo acts both peripherally and centrally through mu opioid receptors to reduce pain perception.

Reduction of voltage gated sodium channel protein in DRG by vector mediated miRNA reduces pain in rats with painful diabetic neuropathy

BackgroundPainful neuropathy is a common complication of diabetes. Previous studies have identified significant increases in the amount of voltage gated sodium channel isoforms NaV1.7 and NaV1.3 protein in the dorsal root ganglia (DRG) of rats with streptozotocin (STZ)-induced diabetes. We found that gene transfer-mediated release of the inhibitory neurotransmitters enkephalin or gamma amino butyric acid (GABA) from DRG neurons in diabetic animals reduced pain-related behaviors coincident with a reduction in NaV1.7 protein levels in DRG in vivo. To further evaluate the role of NaVα subunit levels in DRG in the pathogenesis of pain in diabetic neuropathy, we constructed a non-replicating herpes simplex virus (HSV)-based vector expressing a microRNA (miRNA) against NaVα subunits.ResultsSubcutaneous inoculation of the miRNA-expressing HSV vector into the feet of diabetic rats to transduce DRG resulted in a reduction in NaVα subunit levels in DRG neurons, coincident with a reduction in cold allodynia, thermal hyperalgesia and mechanical hyperalgesia.ConclusionsThese data support the role of increased NaVα protein in DRG in the pathogenesis of pain in diabetic neuropathy, and provide a proof-of-principle demonstration for the development of a novel therapy that could be used to treat intractable pain in patients with diabetic neuropathy.

The Role of TNFα in the Periaqueductal Gray During Naloxone-Precipitated Morphine Withdrawal in Rats

Tolerance and dependence are common complications of long-term treatment of pain with opioids, which substantially limit the long-term use of these drugs. The mechanisms underlying these phenomena are poorly understood. Studies have implicated the midbrain periaqueductal gray (PAG) in the pathogenesis of morphine withdrawal, and recent evidence suggests that proinflammatory cytokines in the PAG may play an important role in morphine withdrawal. Here we report that chronic morphine withdrawal-induced upregulation of glial fibrillary acidic protein (GFAP), tumor necrosis factor alpha (TNFα) and phosphorylation of ERK1/2 (pERK1/2) in the caudal ventrolateral PAG (vlPAG). Microinjection of recombinant TNFα into the vlPAG followed by intraperitoneal naloxone resulted in morphine withdrawal-like behavioral signs, and upregulation of pERK1/2, expression of Fos, and phosphorylation of cAMP response element binding (pCREB) protein. We used a herpes simplex virus (HSV)-based vector expressing p55 soluble TNF receptor (sTNFR) microinjected into the PAG to examine the role of the proinflammatory cytokine TNFα in the PAG in the naloxone-precipitated withdrawal response. Microinjection of HSV vector expressing sTNFR into the PAG before the start of morphine treatment significantly reduced the naloxone-precipitated withdrawal behavioral response and downregulated the expression of GFAP and TNFα in astrocytes of the PAG. TNFR type I colocalized with neuronal pERK1/2. Microinjection of HSV vector expressing sTNFR into the PAG also significantly reduced the phosphorylation of both ERK1/2 and CREB, and reduced Fos immunoreactivity in neurons of the PAG following naloxone-precipitated withdrawal. These results support the concept that proinflammatory cytokines expressed in astrocytes in the PAG may play an important role in the pathogenesis of morphine withdrawal response.